Monomers for preparation of oligonucleotides having chiral phosphorus linkages

ABSTRACT

Sequence-specific oligonucleotides are provided having substantially pure chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp phosphotriester, and chiral Rp phosphotriester linkages. The novel oligonucleotides are prepared via a stereospecific SN 2  nucleophilic attack of a phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate anion on the 3&#39; position of a xylonucleotide. The reaction proceeds via inversion at the 3&#39; position of the xylo reactant species, resulting in the incorporation of phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate linked ribofuranosyl sugar moieties into the oligonucleotide.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No. PCT/US92/08797, filed Oct. 14, 1992, which is a continuation-in-part ofapplication Ser. No. 463,358 filed Jan. 11, 1990, now abandoned and ofapplication Ser. No. 566,977, filed Aug. 13, 1990, now abandoned. Theentire disclosures of both applications, which are assigned to theassignee of this invention, are incorporated herein by reference.

FIELD OF THE INVENTION

This invention is directed to sequence-specific oligonucleotides havingchiral phosphorus linkages and to a novel chemical synthesis of theseand other oligonucleotides. The invention includes chiralalkylphosphonate, chiral phosphotriester, and chiralphosphoramidate-linked oligonucleotides. The invention further includeschiral phosphorothioate, chiral alkylphosphonate, chiralphosphotriester, and chiral phosphoramidate-linked oligonucleotides thatcontain at least one modified nucleoside unit. The novel chemicalsynthesis provides such chiral phosphorothioate, chiralalkylphosphonate, chiral phosphotriester, and chiral phosphoramidateoligonucleotides as well as "natural" or "wild type" phosphodiesteroligonucleotides.

BACKGROUND OF THE INVENTION

Messenger RNA (mRNA) directs protein synthesis. As a therapeuticstrategy, antisense therapy strives to disrupt the synthesis of targetproteins by using a sequence-specific oligonucleotide to form a stableheteroduplex with its corresponding mRNA. Such antisenseoligonucleotides generally have been natural phosphodiesteroligonucleotides.

As contrasted to natural phosphodiester oligonucleotides, the use ofphosphorothioate, methylphosphonate, phosphotriester or phosphoramidateoligonucleotides in antisense therapy provides certain distinguishingfeatures. Each of the phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate phosphorus linkages can exist asdiastereomers. Certain of these phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate oligonucleotides have a greaterresistance to nucleases. Some have solubilities similar to thesolubility of natural phosphodiester oligonucleotides. Other havesolubilities different from that of the natural phosphodiesteroligonucleotides. Some are generally more chemically orthermodynamically stable than the natural phosphodiesteroligonucleotides. At least the phosphorothioates haveoligonucleotide-RNA heteroduplexes that can serve as substrates forendogenous RNase H.

The phosphorothioate oligonucleotides, like the natural phosphodiesteroligonucleotides, are soluble in aqueous media. In contrast,methylphosphonate, phosphotriester, and phosphoramidateoligonucleotides, which lack a charge on the phosphorus group, canpenetrate cell membranes to a greater extent and, thus, facilitatecellular uptake. The internucleotide linkage in methylphosphonateoligonucleotides is more base-labile than that of the naturalphosphodiester internucleotide linkage, while the internucleotidelinkage of the phosphorothioate oligonucleotides is more stable than thenatural phosphodiester oligonucleotide linkage.

The resistance of phosphorothioate oligonucleotides to nucleases hasbeen demonstrated by their long half-life in the presence of variousnucleases relative to natural phosphodiester oligonucleotides. Thisresistance to nucleolytic degradation in vitro also applies to in vivodegradation by endogenous nucleases. This in vivo stability has beenattributed to the inability of 3'-5' plasma exonucleases to degrade sucholigonucleotides. Phosphotriester and methylphosphonate oligonucleotidesalso are resistant to nuclease degradation, while phosphoramidateoligonucleotides show some sequence dependency.

Since they exist as diastereomers, phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate oligonucleotides synthesized usingknown, automated techniques result in racemic mixtures of Rp and Spdiastereomers at the individual phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate linkages. Thus, a 15-meroligonucleotide containing 14 asymmetric linkages has 2¹⁴, i.e. 16,384,possible stereoisomers. Accordingly, it is possible that only a smallpercentage of the oligonucleotides in a racemic mixture will hybridizeto a target mRNA or DNA with sufficient affinity to prove useful inantisense or probe technology.

Miller, P.S., McParland, K.B., Jayaraman, K., and Ts'o, P.O.P (1981),Biochemistry, 20:1874, found that small di-, tri- andtetramethylphosphonate and phosphotriester oligonucleotides hybridize tounmodified strands with greater affinity than natural phosphodiesteroligonucleotides. Similar increased hybridization was noted for smallphosphotriester and phosphoramidate oligonucleotides; Koole, L.H., vanGenderen, M.H.P., Reiners, R.G., and Buck, H.M. (1987), Proc. K. Ned.Adad. Wet., 90:41; Letsinger, R.L., Bach, S.A., and Eadie, J.S. (1986),Nucleic Acids Res., 14:3487; and Jager, A., Levy, M.J., and Hecht, S.M.(1988), Biochemistry, 27:7237. The effects of the racemic diastereomerson hybridization becomes even more complex as chain length increases.

Bryant, F.R. and Benkovic, S.J. (1979), Biochemistry, 18:2825 studiedthe effects of diesterase on the diastereomers of ATP. Published patentapplication PCT/US88/03634 discloses dimers and trimers of 2'-5'- linkeddiastereomeric adenosine units. Niewiarowski, W., Lesnikowski, Z.J.,Wilk, A., Guga, P., Okruszek, A., Uznanski, B., and Stec, W. (1987),Acta Biochimica Polonia, 34:217, synthesized diastereomeric dimers ofthymidine, as did Fujii, M., Ozaki, K., Sekine, M., and Hata, T. (1987),Tetrahedron, 43:3395.

Stec, W.J., Zon, G., and Uznanski, B. (1985), J. Chromatography,326:263, have reported the synthesis of certain racemic mixtures ofphosphorothioate or methyphosphonate oligonucleotides. However, theywere only able to resolve the diastereomers of certain small oligomershaving one or two diastereomeric phosphorus linkages.

In a preliminary report, J. W. Stec, Oligonucleotides as antisenseinhibitors of gene expression: Therapeutic implications, meetingabstracts, Jun. 18-21, 1989, noted that a non-sequence-specificthymidine homopolymer octomer--i.e. a (dT)₈ -mer, having"all-except-one" Rp configuration methylphosphonate linkages--formed athermodynamically more stable hybrid with a 15-mer deoxyadenosinehomopolymer--i.e. a d(A)₁₅ -mer--than did a similar thymidinehomopolymer having "all-except-one" Sp configuration methylphosphonatelinkages. The hybrid between the "all-except-one" Rp (dT)₈ -mer and thed(A)₁₅ -mer had a Tm of 38° C. while the Tm of the "all-except-one" Sp(dT)₈ -mer and the d(A)₁₅ -mer was <0° C. The hybrid between a (dT)₈-mer having natural phosphodiester linkages, i.e. octathymidylic acid,and the d(A)₁₅ -mer was reported to have a Tm of 14° C. Theall-except-one"thymidine homopolymer octamers were formed from twothymidine methylphosphonate tetrameric diastereomers linked by a naturalphosphodiester linkage.

To date, it has not been possible to chemically synthesize anoligonucleotide having more than two adjacent, chirally pure phosphorouslinkages. Indeed, even in homopolymers it has been possible to produceonly three such adjacent chiral linkages. For an oligonucleotide to beuseful as an antisense compound, many nucleotides must be present. Whilenot wishing to be bound by any particular theory, it is presentlybelieved that generally at least about 10 or more nucleotides arenecessary for an oligonucleotide to be of optimal use as an antisensecompound. Because it has not been possible to resolve more than two orthree adjacent phosphorus linkages, the effects of induced chirality inthe phosphorus linkages of chemically synthesized antisenseoligonucleotides has not been well assessed heretofore.

Except as noted above, the sequence-specific phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate oligonucleotidesobtained utilizing known automated synthetic techniques have beenracemic mixtures. Indeed, it was recently stated in a review articlethat: "It is not yet possible to synthesize by chemical meansdiastereomerically pure chains of the length necessary for antisenseinhibition," see J. Goodchild (1990) Bioconjugate Chemistry, 1:165.

The use of enzymatic methods to synthesize oligonucleotides havingchiral phosphorous linkages has also been investigated. Burgers, P.M.J.and Eckstein, F. (1979), J. Biological Chemistry, 254:6889; and Gupta,A., DeBrosse, C., and Benkovic, S.J. (1982) J. Bio. Chem., 256:7689enzymatically synthesized diastereomeric polydeoxyadenylic acid havingphosphorothioate linkages. Brody, R.S. and Frey, P.S. (1981),Biochemistry, 20:1245; Eckstein, F. and Jovin, T.M. (1983),Biochemistry, 2:4546; Brody, R.S., Adler, S., Modrich, P., Stec, W.J.,Leznikowski, Z.J., and Frey, P.A. (1982) Biochemistry, 21: 2570-2572;and Romaniuk, P.J. and Eckstein, F. (1982) J. Biol. Chem., 257:7684-7688all enzymatically synthesized poly TpA and poly ApT phosphorothioateswhile Burgers, P.M.J. and Eckstein, F. (1978) Proc. Natl. Acad. Sci.USA, 75: 4798-4800 enzymatically synthesized poly UpA phosphorothioates.Cruse, W.B.T., Salisbury, T., Brown, T., Cosstick, R. Eckstein, F., andKennard, O. (1986), J. Mol. Biol., 192:891, linked three diastereomericRp GpC phosphorothioate dimers via natural phosphodiester bonds into ahexamer. Most recently Ueda, T., Tohda, H., Chikazuni, N., Eckstein, R.,and Watanabe, K. (1991) Nucleic Acids Research, 19:547, enzymaticallysynthesized RNA's having from several hundred to ten thousandnucleotides incorporating Rp diastereomeric phosphorothioate linkages.Enzymatic synthesis, however, depends on the availability of suitablepolymerases that may or may not be available, especially for modifiednucleoside precursors.

Thus, while phosphorothioate, alkylphosphonate, phosphoamidate, andphosphotriester oligonucleotides have useful characteristics, little isknown concerning the effects of differing chirality at the phosphoruslinkages. It would therefore be of great advantage to provideoligonucleotides having phosphorous linkages of controlledstereochemistry.

OBJECTS OF THE INVENTION

Accordingly, it is one object of this invention to providesequence-specific oligonucleotides having chirally purephosphorothioate, alkylphosphonate, phosphotriester or phosphoramidatelinkages.

It is a further object to provide phosphorothioate, alkylphosphonate,phosphoramidate, and phosphotriester oligonucleotides comprisingsubstantially all Rp or all Sp linkages.

It is another object to provide phosphorothioate, alkylphosphonate,phosphoramidate, and phosphotriester oligonucleotides that haveantisense hybridizability against DNA and RNA sequences.

It is still another object of this invention to providephosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriesteroligonucleotides for use in antisense diagnostics and therapeutics.

A further object is to provide research and diagnostic methods andmaterials for assaying bodily states in animals, especially diseasedstates.

Another object is to provide therapeutic and research methods andmaterials for the treatment of diseases through modulation of theactivity of DNA and RNA.

It is yet another object to provide new methods for synthesizingsequence-specific oligonucleotides having chirally purephosphorothioate, methylphosphonate, phosphotriester or phosphoramidatelinkages.

SUMMARY OF THE INVENTION

The present invention provides stereoselective methods for preparingsequence-specific oligonucleotides having chiral phosphorous linkages.In certain preferred embodiments, these methods comprise the steps of:

(a) selecting a first synthon having structure (1): ##STR1## wherein Qis O or CH₂ ;

R_(A) and R_(B) are H, lower alkyl, substituted lower alkyl, an RNACleaving moiety, a group which improves the pharmacokinetic propertiesof an oligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide;

R_(D) is O, S, methyl, alkoxy, thioalkoxy, amino or substituted amino;

R_(E) is O or S;

R_(X) is H, OH, or a sugar derivatizing group;

R_(X) is a naturally occurring or synthetic nucleoside base or blockednucleoside base; and

Y is a stable blocking group, a solid state support, a nucleotide on asolid state support, or an oligonucleotide on a solid state support;

(b) selecting a second synthon having structure (2): ##STR2## whereinR_(F) is a labile blocking group; and

L is a leaving group or together L and B_(X) are a 2-3' or 6-3'pyrimidine or 8-3' purine cyclonucleoside;

(c) adding the second synthon to the first synthon in the presence of abase to effect nucleophilic attack of the 5'-phosphate of the firstsynthon at the 3'-position of the second synthon to yield a new firstsynthon having structure (3): ##STR3## via a stereospecific inversion ofconfiguration at the 3' position of the second synthon; and

(d) treating the new first synthon with a reagent to remove the labileblocking group R_(F).

Additional nucleotides are added to the new first synthon by repeatingsteps (b), (c), and (d) for each additional nucleotide. Preferably,R_(F) is an acid-labile blocking group and said new first synthon instep (d) is treated with an acidic reagent to remove said acid-labileR_(F) blocking group.

The present invention also provides sequence-specific oligonucleotidescomprising a plurality of nucleotides linked by chiralphosphorothioate,methylphosphonate, phosphotriester or phosphoramidate oligonucleotideslinkages wherein at least one of the nucleosides is a non-naturallyoccurring nucleoside. Preferably, the nucleosides are connected vialinkages selected from the group consisting of chiral Spphosphorothioate, chiral Rp phosphorothioate, chiral Spalkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate,chiral Rp phosphoamidate, chiral Sp chiral phosphotriester or chiral Rpphosphotriester linkages. In one embodiment of the invention each of thelinkages of the oligonucleotide is a substantially pure chiralphosphorous linkage. In other embodiments less than all of the phosphatelinkages are substantially pure chiral phosphorous linkages. In furtherembodiments, the oligonucleotides of the invention form at least aportion of a targeted RNA or DNA sequence.

The present invention also provides oligonucleotides comprisingnucleoside units joined together by either all Sp phosphotriesterlinkages, all Rp phosphotriester linkages, all Sp phosphoramidatelinkages, or all Rp phosphoramidate linkages. Also provided areoligonucleotides having at least 10 nucleoside units joined together byeither all Sp alkylphosphonate linkages or all Rp alkylphosphonatelinkages. Preferably such alkylphosphonate linkages aremethylphosphonate linkages. Each of these oligonucleotides can form atleast a portion of a targeted RNA or DNA sequence.

In preferred embodiments of the invention, the oligonucleotides includenon-naturally occurring nucleoside units incorporated into theoligonucleotide chain. Such nucleoside units preferably have structure(4) or structure (5): ##STR4## wherein Q, R_(B), R_(G), and B_(X) aredefined as above and R_(C) is H, OH, lower alkyl, substituted loweralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl,S-alkyl, substituted S-alkyl, SOMe, SO₂ Me, ONO₂, NO₂, N₃, NH₂,NH-alkyl, substituted NH-alkyl, OCH₂ CH═CH₂, OCH═CH₂, OCH₂ CCH, OCCH,aralkyl, heteroaralkyl, heterocycloalkyl, poly-alkylamino, substitutedsilyl, an RNA cleaving moiety, a group which improves thepharmacodynamic properties of an oligonucleotide, or a group whichimproves the pharmacokinetic properties of an oligonucleotide.

In preferred embodiments, B_(X) is a pyrimidinyl-1 or purinyl-9 moietysuch as, for example, adenine, guanine, hypoxanthine, uracil, thymine,cytosine, 2-aminoadenine or 5-methylcytosine.

In further preferred embodiments, the modified nucleosides includesnucleosides having structures (6)-(11): ##STR5## wherein: G and K are,independently, C or N;

J is N or CR₂ R₃ ;

R₁ is OH or NH₂ ;

R₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, loweralkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino,heterocycloalkyl, heterocycloalkylamino, aminoalkylamino,hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a groupwhich improves the pharmacokinetic properties of an oligonucleotide, ora group which improves the pharmacodynamic properties of anoligonucleotide;

R₄ and R₅ are, independently, H, OH, NH₂, lower alkyl, substituted loweralkyl, substituted amino, an RNA cleaving moiety, a group which improvesthe pharmacokinetic properties of an oligonucleotide, or a group whichimproves the pharmacodynamic properties of an oligonucleotide;

R₆ and R₇ are, independently, H, OH, NH₂, SH, halogen, CONH₂, C(NH)NH₂,C(O)O-alkyl, CSNH₂, CN, C(NH)NHOH, lower alkyl, substituted lower alkyl,substituted amino, an RNA cleaving moiety, a group which improves thepharmacokinetic properties of an oligonucleotide, or a group whichimproves the pharmacodynamic properties of an oligonucleotide; and

X is a sugar or a sugar analog moiety where said sugar analog moiety isa sugar substituted with at least one substituent comprising an RNAcleaving moiety, a group which improves the pharmacodynamic propertiesof an oligonucleotide, or a group which improves the pharmacokineticproperties of an oligonucleotide.

The present invention also provides compounds which are useful informing the oligonucleotides of invention. Such compounds have structure(12): ##STR6## wherein Q, R_(A), R_(D), R_(E), R_(X), L, and B_(X) aredefined as above and R_(F) is H or a labile blocking group.

The oligonucleotides of the invention are useful to increase thethermodynamic stability of heteroduplexes with target RNA and DNA.Certain of the oligonucleotides of the invention are useful to elicitRNase H activity as a termination event. Certain other oligonucleotidesare useful to increase nuclease resistance. The oligonucleotides of theinvention are also useful to test for antisense activity using reportergenes in suitable assays and to test antisense activity against selectedcellular target mRNA's in cultured cells.

DETAILED DESCRIPTION OF THE INVENTION

As will be recognized, adjacent nucleosides of a naturally occurring orwild type oligonucleotide are joined together by phosphodiesterlinkages, i.e. diesters of phosphoric acid. The natural phosphodiesterlinkages in oligonucleotides are at the same time both non-chiral andprochiral sites. Substitution of one of the oxygen atoms of thephosphate moiety of a nucleotide with another atom yields an asymmetriccenter on the phosphorus atom. Since a nucleotide unit already containsa first asymmetrical center within its sugar moiety, further asymmetryat the phosphorus atom of the nucleotide yields a diasymmetricnucleotide. Such a diasymmetric nucleotide is a chiral nucleotide havingSp and Rp diastereomers.

Substitution of one of the oxygen atoms of the phosphate moiety of anucleotide with a sulfur atom yields Sp and Rp diastereomericphosphorothioate analogs. Similarly, substitution of a phosphate oxygenatom by an alkyl moiety yields diastereomeric alkylphosphonate analogs.Substitution with an alkoxy group yields diastereomeric Sp and Rpphosphotriesters. Substitution with a thioalkoxy group yields a mixedtriester--a phosphodiesterthioester. Substitution with an amine or asubstituted amine (including heterocyclic amines) yields diastereomericSp and Rp phosphoramidates.

It will be appreciated that the terms phosphate" and "phosphate anion"as employed in connection with the present invention include nucleotidesand oligonucleotides derived by replacement of one of the oxygen atomsof a naturally occurring phosphate moiety with a heteroatom, an alkylgroup or an alkyoxy group. Thus, the terms "phosphate" or "phosphateanion" include naturally occurring nucleotides, phosphodiesters ofnaturally occurring oligonucleotides, as well as phosphorothioate,alkylphosphonate, phosphotriester, and phosphoamidate oligonucleotides.

Since there exist numerous phosphodiester linkages in anoligonucleotide, substitution of an oxygen atom by another atom such as,for example, sulfur, nitrogen, or carbon in one or more of the phosphatemoieties yields a racemic mixture unless such substitution occurs in astereospecific manner. As a practical matter, see Stec, W.J., Zon, G.,and Uznanski, B. (1985), J. Chromatography, 326:263, above. Separationof the diastereomers of racemic mixtures of non-stereospecificsynthesized oligonucleotides is only possible when there are a minimumof diasymmetric sites, for example, two diasymmetric sites. Since thediasymmetric substituent group at each diastereomeric phosphorus atomcould have steric, ionic or other effects on conformation, binding, andthe like at each such site, sequence-specific oligonucleotides havingall Sp or all Rp chiral phosphorus linkages are desirable.

In accordance with this invention, sequence-specific oligonucleotidesare provided comprising substantially pure chiral phosphate linkagessuch as, for example, phosphorothioate, methylphosphonate,phosphotriesteror phosphoramidate linkages. In contrast to prior artsynthetic oligonucleotides, at least certain of the chiral phosphorouslinkages of the present oligonucleotides are not racemic in nature but,rather, possess relatively high enantiomeric purity. As will berecognized by those skilled in the art, enantiomeric purity --also knownas chiral purity--is manifested for a chemical compound by thepredominance of one enantiomer over the other. Thus, an oligonucleotidecan be said to possess a substantially pure chiral phosphate linkagewhere, for example, the Sp form of that linkage greatly predominatesover the Rp form. In accordance with the present invention, at leastcertain of the chiral phosphate linkages present in an oligonucleotideshould have chiral purity greater than about 75%. Preferably suchlinkages have chiral purity greater than about 90%, more preferablygreater than about 95%, even more preferably about 100%. Chiral puritymay be determined by any of the many methods known in the art, includingbut not limited to x-ray diffraction, optical rotary dispersion, andcircular dichroism.

The oligonucleotides of the invention are expected to exhibit one ormore efficacious properties such as, for example, hybridization withtargeted RNA's and DNA's, cellular absorption and transport, or improvedenzymatic interaction. At the same time, it is expected that theseimprovements to the basic oligonucleotide sequences will notsignificantly diminish existing properties of the basic oligonucleotidesequence. Thus, the present improvements are likely to lead to improveddrugs, diagnostics, and research reagents.

Further improvements likely can be effected by making one or moresubstitutions or modifications to the base or the sugar moieties of theindividual nucleosides employed to prepare the chiral oligonucleotidesof the invention. Such substitutions or modifications generally comprisederivation at a site on the nucleoside base or at a site on thenucleoside sugar, provided such derivation does not interfere with thestereoselective syntheses of the present invention by, for example,blocking nucleophilic attack of the 5'-phosphate of a first synthon atthe 3'-position of a second synthon. In certain embodiments, one or moreof the nucleosides of the chiral oligonucleotides of the inventioninclude a naturally occurring nucleoside unit which has been substitutedor modified. These non-naturally occurring or "modified" nucleosideunits preferably have either structure (4) or structure (5): ##STR7##wherein: Q is O or CHR_(G) ;

R_(A) and R_(B) are H, lower alkyl, substituted lower alkyl, an RNAcleaving moiety, a group which improves the pharmacokinetic propertiesof an oligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide;

R_(C) is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN,CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substitutedS-alkyl, SOMe, SO₂ Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substitutedNH-alkyl, OCH₂ CH═CH₂, OCH═CH₂, OCH₂ CCH, OCCH, aralkyl, heteroaralkyl,heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleavingmoiety, a group which improves the pharmacodynamic properties of anoligonucleotide, or a group which improves the pharmacokineticproperties of an oligonucleotide;

R_(G) is H, lower alkyl, substituted lower alkyl, an RNA cleavingmoiety, a group which improves the pharmacokinetic properties of anoligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide; and

B_(X) is a nucleoside base, a blocked nucleoside base, a nucleoside baseanalog, or a blocked nucleoside base analog.

In preferred embodiments B_(X) is a pyrimidinyl-1 or purinyl-9 moiety asfor instance adenine, guanine, hypoxanthine, uracil, thymine, cytosine,2-aminoadenine or 5-methylcytosine. Preferably, B_(X) is selected suchthat a modified nucleoside has one of the structures (6)-(11): ##STR8##wherein: G and K are, independently, CH or N;

J is N or CR₂ R₃ ;

R₁ is OH or NH₂ ;

R₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, loweralkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino,heterocycloalkyl, heterocycloalkylamino, aminoalkylamino,hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a groupwhich improves the pharmacokinetic properties of an oligonucleotide, ora group which improves the pharmacodynamic properties of anoligonucleotide;

R₄ and R₅ are, independently, H, OH, NH₂, lower alkyl, substituted loweralkyl, substituted amino, an RNA cleaving moiety, a group which improvesthe pharmacokinetic properties of an oligonucleotide, or a group whichimproves the pharmacodynamic properties of an oligonucleotide;

R₆ and R₇ are, independently, H, OH, NH₂, SH, halogen, CONH₂, C(NH)NH₂,C(O)O-alkyl, CSNH₂, CN, C(NH)NHOH, lower alkyl, substituted lower alkyl,substituted amino, an RNA cleaving moiety, a group which improves thepharmacokinetic properties of an oligonucleotide, or a group whichimproves the pharmacodynamic properties of an oligonucleotide; and

X is a sugar or a sugar analog moiety where said sugar analog moiety isa sugar substituted with at least one substituent comprising an RNAcleaving moiety, a group which improves the pharmacodynamic propertiesof an oligonucleotide, or a group which improves the pharmacokineticproperties of an oligonucleotide. It is preferred that X have thegeneral structure (4) or (5).

For the purposes of this invention, improving pharmacodynamic propertiesmeans improving oligonucleotide uptake, enhanced oligonucleotideresistance to degradation, and/or strengthened sequence-specifichybridization with RNA and improving pharmacokinetic properties meansimproved oligonucleotide uptake, distribution, metabolism or excretion.RNA cleaving moieties are chemical compounds or residues which are ableto cleave an RNA strand in either a random or, preferably, asequence-specific fashion.

Exemplary base moieties of the invention are any of the naturalpyrimidinyl-1- or purinyl-9- bases including uracil, thymine, cytosine,adenine, guanine, 5-alkylcytosines such as 5-methylcytosine,hypoxanthine, 2-aminoadenine, and other modified bases as depicted inthe formulas above. Exemplary sugars include ribofuranosyl,2'-deoxyribofuranosyl, their corresponding five membered ringcarbocyclic analogs as well as other modified sugars depicted in theformulas above. Particularly preferred modified sugars include 2'-fluoroand 2'-O-methyl-2'-deoxyribofuranosyl, i.e. 2'-fluoro and2'-O-methyl-β-D-erythro-pentofuranosyl.

Alkyl groups of the invention include but are not limited to C₁ -C₁₂straight and branched chained alkyls such as methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,isopropyl, 2-butyl, isobutyl, 2-methylbutyl, isopentyl, 2-methyl-pentyl,3-methylpentyl, 2-ethylhexyl, 2-propylpentyl. Alkenyl groups include butare not limited to unsaturated moieties derived from the above alkylgroups including but not limited to vinyl, allyl, crotyl, propargyl.Aryl groups include but are not limited to phenyl, tolyl, benzyl,naphthyl, anthracyl, phenanthryl, and xylyl. Halogens include fluorine,chlorine, bromine, and iodine. Suitable heterocyclic groups include butare not limited to imidazole, tetrazole, triazole, pyrrolidine,piperidine, piperazine, and morpholine. Carbocyclic groups include 3, 4,5, and 6-membered substituted and unsubstituted alkyl and alkenylcarbocyclic rings. Amines include amines of all of the above alkyl,alkenyl, and aryl groups including primary and secondary amines and"masked amines" such as phthalimide. Amines are also meant to includepolyalkylamino compounds and aminoalkylamines such as aminopropylamineand further heterocyclo-alkylamines such as imidazol-1, 2 or4-yl-propylamine. Substituent groups for the above include but are notlimited to other alkyl, haloalkyl, alkenyl, alkoxy, thioalkoxy,haloalkoxy, and aryl groups as well as halogen, hydroxyl, amino, azido,carboxy, cyano, nitro, mercapto, sulfides, sulfones, and sulfoxides.Other suitable substituent groups also include rhodamines, coumarins,acridones, pyrenes, stilbenes, oxazolo-pryidocarbazoles, anthraquinones,phenanthridines, phenazines, azidobenzenes, psoralens, porphyrins,cholesterols, and other "conjugate" groups.

Methods of synthesizing such modified nucleosides are set forth incopending applications for United States Letters Patent, assigned to theassignee of this invention, and entitled Compositions and Methods forModulating RNA Activity, Ser. No. 463,358, filed Jan 11, 1990; SugarModified Oligonucleotides That Detect And Modulate Gene Expression, Ser.No. 566,977, filed Aug. 13,1990; and Compositions and Methods forModulating RNA Activity, Ser. No. US92/08797, filed Jan. 11, 1991, theentire disclosures of which are incorporated herein by reference.

The chirally pure phosphorothioate, methylphosphonate, phosphotriesteror phosphoramidate oligonucleotides of the invention can be evaluatedfor their ability to act as inhibitors of RNA translation in vivo.Various therapeutic areas can be targeted for such antisense potential.These therapeutic areas include but are not limited to herpes virus(HSV), the TAR and tat regions of HIV, the codon regions of Candidaalbicans chitin synthetase and Candida albicans B tubulin, papillomavirus (HPV), the ras oncogene and protooncogene, ICAM-1 (intercellularadhesion molecule-1) cytokine, and 5'-lipoxygenase. A targeted regionfor HSV includes GTC CGC GTC CAT GTC GGC. A targeted region for HIVincludes GCT CCC AGG CTC AGA TCT. A targeted region for Candida albicansincludes TGT CGA TAA TAT TAC CA. A targeted region for humanpapillomavirus, e.g. virus types HPV-11 and HPV-18, includes TTG CTT CCATCT TCC TCG TC. A targeted region for ras includes TCC GTC ATC GCT CCTCAG GG. A targeted region for ICAM-1 includes TGG GAG CCA TAG CGA GGCand the sequence CGA CTA TGC AAG TAC is a useful target sequence for5-lipoxygenase. In each of the above sequences the individual nucleotideunits of the oligonucleotides are listed in a 5' to 3' sense from leftto right.

The phosphorothioate, methylphosphonate, phosphotriester orphosphoramidate oligonucleotides of the invention may be used intherapeutics, as diagnostics, and for research, as specified in thefollowing copending applications for United States Letters Patentassigned to the assignee of this invention: Compositions and Methods forModulating RNA Activity, Ser. No. 463,358, filed 1/11/90; AntisenseOligonucleotide Inhibitors of Papilloma Virus, Ser. No. 445,196 Filed12/4/89; Oligonucleotide Therapies for Modulating the Effects ofHerpesvirus, Ser. No. 485,297, filed 2/26/90; Reagents and Methods forModulating Gene Expression Through RNA Mimicry Ser. No. 497,090, filed3/21/90; Oligonucleotide Modulation of Lipid Metabolism, Ser. No.516,969, filed 4/30/90; Oligonucleotides for Modulating the Effects ofCytomegalovirus Infections, Ser. No. 568,366, filed Aug. 16, 1990;Antisense Inhibitors of the Human Immunodeficiency Virus, Ser. No.521,907, filed 5/11/90; Nuclease Resistant Pyrimidine ModifiedOligonucleotides for Modulation of Gene Expression, Ser. No.558,806,filed 7/27/90; Novel Polyamine Conjugated Oligonucleotides, Ser. No.558,663, filed 7/27/90, now U.S. Pat. No. 5,138,045; Modulation of GeneExpression Through Interference with RNA Secondary Structure, Ser. No.518,929, filed 5/4/90; Oligonucleotide Modulation of Cell Adhesion, Ser.No. 567,286, filed 8/14/90; Inhibition of Influenza Viruses, Ser. No.567,287, filed 8/14/90; Inhibition of Candida, Ser. No. 568,672, filed8/16/90; and Antisense Oligonucleotide Inhibitors of Papillomavirus,Ser. No. PCT/US90/07067, filed 12/3/90. These patents disclose a numberof means whereby improved modulation of RNA and DNA activity may beaccomplished through oligonucleotide interaction. To the extent that thespecific sequences disclosed therein may be used in conjunction with thepresent invention, the disclosures of the foregoing United States patentapplications are incorporated herein by reference.

The oligonucleotides of the invention preferably are prepared via theprocess shown in Scheme 1, wherein a selected nucleotide is coupled toanother nucleotide or to a growing oligonucleotide chain via anucleophilic displacement reaction. As will be recognized, this processis also applicable to the preparation of oligonucleotides comprisingnon-chiral phosphodiester linkages. In Scheme 1, R_(F) is a phosphateblocking group, B_(X) is a suitable heterocyclic nucleoside base(blocked or unblocked), R_(X) is a sugar derivatizing group and Q is anoxygen or methylene group. Together R_(D) and R_(E) are the necessaryoxygen, sulfur, nitrogen, substituted nitrogen, alkoxy or thioalkoxygroups that form the phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate linking groups. For Scheme 1, the Ygroup of the above formulas is depicted as a CPG (Controlled Pore Glass)group. Other Y groups also may be used. ##STR9##

In Scheme 1, a first nucleotide (13) is attached to a o solid state CPGsupport via its 3'-hydroxyl group in a standard manner, yieldingcompound (14). Compound (14), which forms a first synthon, is treatedwith an appropriate base, producing anionic compound (15). Compound (15)is reacted with a first unit of compound (2)--a xylofuranosyl nucleotidebearing blocking group R_(F) on its phosphate functionality and leavinggroup L at its 3' position. Compound (2) is a second synthon. The B_(X)moiety of structure (2) may be the same as or different than the B_(X)moiety of compound (15), depending on the sequence of the desiredoligonucleotide. The anion of compound (15) displaces the leaving groupat the 3' position of compound (2). The displacement proceeds viainversion at the 3' position of the second synthon, forming compound(16) with n=1.

The resulting phosphorothioate, methylphosphonate, phosphotriester orphosphoramidate linkage of compound (16) extends from the 5' position ofthe first synthon (compound (14)) to the 3' position of the secondsynthon (compound (2)). The inversion at the 3' position of the secondsynthon results in the final configuration at the 3' nucleotide derivedfrom the second synthon being a normal ribofuranosyl sugar conformation.Compound (16) on the solid state CPG support is now washed to free it ofany unreacted compound (2).

The second synthon carries a phosphate blocking group R_(F) on itsphosphorothioate, methylphosphonate, phosphotriester or phosphoramidatephosphorus group. After coupling of the second synthon to the firstsynthon to yield compound (16) wherein n=1 and washing, the phosphateblocking group R_(F) is removed with an acid, yielding compound (17)wherein n=1. Compound (17), which represents a new first synthon, is nowtreated with base to generate a further anionic, compound (18) with n=1.Compound (18) is suitable for nucleophilic attack on a further unit ofcompound (2) (the second synthon) to form a new compound (16) whereinn=2. In this further unit having compound (2), the B_(X) moiety may bethe same or different from the B_(X) moiety of either of the nucleotidesof compound (16) wherein n=1, depending on the desired sequence.

Compound (16) wherein n=2 is washed and then treated with acid todeblock the R_(F) blocking group, yielding a further new first synthon,compound (17) wherein n=2. This new first synthon, is now ready to befurther cycled by treatment with base to yield compound (18) whereinn=2, which is now reacted with a further unit having compound (2) toyield a further unit of having structure (16) wherein n=3. Again, B_(X)may be the same or different than previously B_(X) moieties. The cycleis repeated for as many times as necessary to introduced furthernucleotides of the desired sequence via compound (2).

If it is desired to have the 5' terminal end of the finaloligonucleotide as a phosphate group, then the last compound (17) isappropriately removed from the CPG support. If it is desired to have the5' terminal end as a hydroxyl group, then the penultimate nucleotide isadded as compound (22), it is converted to compounds (16), (17), and(18) and the resulting compound (18) is reacted With a xylofuranosylnucleoside, compound (19). Compound (19), like compound (2), includes aleaving group L within its structure. Reaction of compound (19) withcompound (18) yields oligonucleotide, compound (20), which is releasedfrom the CPG support with concentrated ammonium ion to yield the desiredoligonucleotide, compound (21). The ammonium ion treatment will alsoremove any base blocking groups as is standard in automatedoligonucleotide synthesis.

In summary, as shown in Scheme 1, a phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate 5' nucleotide (or the 5'-terminalnucleotide of a growing oligonucleotide) functions as a first synthon.This is converted to an anion with a base. This anion displaces aleaving group at the 3' position of a xylofuranosyl nucleotide. Thexylofuranosyl nucleotide comprises a second synthon. The displacementproceeds via inversion at the 3' position of the second synthon with theresulting phosphorothioate, methylphosphonate, phosphotriester orphosphoramidate linkage that is formed extending from the 5' position ofthe first synthon to the 3' position of the second synthon. Theinversion at the 3' position of the second synthon results in the finalconfiguration at the 3' nucleotide derived from the second synthon beinga normal ribofuranosyl sugar conformation. It has a 3' to 4' transorientations (a ribofuranosyl sugar conformation) that is identical tonatural or wild type oligonucleotides.

The second synthon carries a phosphate blocking group on itsphosphorothioate, methylphosphonate, phosphotriester or phosphoramidatephosphorus group. After coupling of the second synthon to the firstsynthon, this phosphate blocking group is removed, generating a newfirst synthon having an anion at its 5' phosphate suitable fornucleophilic attack on a further second synthon. Thus, after coupling ofthe first and second synthon, the newly joined first and second synthonsnow form a new first synthon. The oligonucleotide is elongatednucleotide by nucleotide via the nucleophilic attack of a phosphateanion at the 5' end of the growing oligonucleotide chain on the leavinggroup at the 3' position of the soon-to-be-added xylofuranosylconfigured second synthon nucleotide.

It is presently preferred that the phosphate blocking group be a basestable, acid labile group. Such a phosphate blocking group maintains thephosphate moiety of the second synthon in a protected form that cannotreact with the leaving group of the second synthon. This inhibitspolymerization of the second synthon during the coupling reaction.

The nucleophilic coupling of the first and second synthons is astereoselective coupling process that maintains the stereospecificconfiguration about the phosphorus atom of the first synthon. Thus theparticular Sp or Rp diastereomeric configuration of a resolvedphosphorothioate, methylphosphonate, phosphotriester or phosphoramidatemoiety at the 5' end of a starting second synthon nucleotide or the 5'terminal end of a growing first synthon oligonucleotide is maintained.While the sugar portion of the second synthon undergoes inversion aboutits 3' position as a result of the coupling process, the phosphateportion of the second synthon retains its stereospecific configuration.After coupling of the second synthon to the first, the phosphate moiety(for example, the phosphorothioate, alkylphosphate, phosphoamidate orphosphotriester moiety) of the second synthon retains its originalstereochemistry. That is, an Rp diastereomeric second synthon retainsthe Rp configuration of its phosphate moiety, while an Sp diastereomericsecond synthon retains the Sp configuration of its phosphate moiety.

For example, to form an Rp chiral phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate oligonucleotide a first Rpdiastereomeric nucleotide is chosen as the first synthon. It is coupledwith an Rp diastereomeric second synthon nucleotide. The resultingdinucleotide maintains the Rp orientation at the inter-nucleotidelinkage between the first and second nucleotides. When a thirdnucleotide is next coupled to the first two, the Rp diastereomericphosphate moiety from the second nucleotide forms the inter-nucleotidelinkage between the second and third nucleotides and is maintained inthe Rp orientation. If the third nucleotide was also an Rpdiastereomeric nucleotide then when a fourth nucleotide is added to thegrowing oligonucleotide chain, the inter-nucleotide linkage betweennucleotides three and four is also an Rp diastereomeric linkage. If eachadded "second synthon" is also an Rp diastereomer, then the resultingoligonucleotide will contain only Rp inter-nucleotide linkages. If anoligonucleotide having Sp inter-nucleotide linkages is desired, then thefirst nucleotide and each of the added subsequent nucleotides areselected as Sp diastereomeric nucleotides.

The first synthon can be a first nucleotide or a growing oligonucleotidechain. If it is desired that each of the nucleotides of theoligonucleotide be ribofuranoside configured nucleotides, then the firstnucleotide is selected as a ribofuranoside configured nucleotide. Eachadded second synthon, while added as a xylofuranoside configurednucleotide, after inversion is converted to a ribofuranoside configurednucleotide.

The 3' position of the first nucleotide is either blocked if a solutionreaction is practiced or is coupled to a solid state support if a solidstate reaction (as for instance one utilizing a DNA synthesizer) ispracticed. Each additional nucleotide of the oligonucleotide is thenderived from a xylofuranosyl nucleotide, i.e. a second synthon. Becausethe first nucleotide of the oligonucleotide can be a "standard"ribofuranosyl nucleotide coupled via its 3' hydroxyl to a solid statesupport, the standard solid state supports known in the art, such ascontrolled pore glass (CPG) supports, can be utilized and the secondsynthons added as reagents to the growing oligonucleotide on a standardDNA synthesizer such as, for example, an Applied Biosystems Inc. 380BNucleic Acid Synthesizer. However, unlike standard DNA synthesizertechniques, nucleotide coupling is not achieved using activatedphosphoamidate chemistry. Instead, the above-noted nucleophilicdisplacement reaction of a phosphate anion on a 3' leaving group of axylofuranosyl nucleotide is utilized as the coupling reaction. Even whenchiral phosphoramidate phosphate linkages are being incorporated intothe sequence-specific chiral oligonucleotides of the invention, thephosphoramidate groups of individual nucleotides are not directly usedto effect the coupling reaction between nucleotides. Rather, as withphosphorothioates, alkylphosphonates, and phosphotriesters, an anion isgenerated at the phosphate moiety. It is this anion --not an activatedamidate species--that is the activated species for effecting coupling.

Once the first nucleotide is loaded on a solid state support utilizingstandard techniques, the anion necessary for nucleophilic attack isgenerated via treatment of the first nucleotide, i.e. the first synthon,with a base. Suitable bases include but are not limited to sodiumhydride, methylmagnesium chloride, and t-butylmagnesium chloride. Theseare used in a suitable solvent such as acetonitrile, tetrahydrofuran ordioxane.

The second synthon is added either concurrently with the base orsubsequent to it. After coupling, the growing oligonucleotide is washedwith a solvent and then treated with a reagent to effect deblocking ofthe phosphate blocking group of the second synthon. If a preferredacid-labile blocking group is used to block the phosphate of the secondsynthon, deblocking is easily effected by treating the growingoligonucleotide on the solid state support with an appropriate acid.

Suitable acid-labile blocking groups for the phosphates of the secondsynthon include but are not limited to t-butyl, dimethoxytrityl (DMT) ortetrahydropyranyl groups. Suitable acids for deblocking the secondsynthon phosphate blocking group include but are not limited to aceticacid, trichloroacetic acid, and trifluoromethane sulfonic acid. Suchacids are suitably soluble in solvents such as tetrahydrofuran,acetonitrile, dioxane, and the like.

Following treatment with an appropriate deblocking reagent to effectdeblocking of the phosphate protecting group, the growingoligonucleotide is then washed with an appropriate solvent such astetrahydrofuran, acetonitrile or dioxane. The oligonucleotide is nowready for the addition of a further nucleotide via treatment with baseto generate an anion on the 5' terminal phosphate followed by theaddition of a further second synthon. Alternatively, the anion can begenerated concurrently with addition of a further second synthon.Suitable leaving groups for inclusion at the 3' position of thexylofuranosyl second synthon include but are not limited to the groupconsisting of halogen, alkylsulfonyl, substituted alkylsulfonyl,arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl ortrichloroacetimidate. A more preferred group includes chloro, fluoro,bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl,methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate),p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate),trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl,imidazolesulfonyl, and 2,4,6-trichlorophenyl. These leaving groups aresubject to SN₂ displacement reactions with inversion about the 3'position of the sugar to provide the required 3'-4' trans ribofuranosylconfiguration after inversion. The ionized oxygen atom of the phosphatemoiety of the first synthon displaces these leaving groups to effect thecoupling of the first synthon to the second synthon.

A further class of leaving groups include certain sugar-basecyclonucleosides. These include 2,3' or 6,3'-cyclopyrimidines and8,3'-cyclopurine nucleosides. These nucleosides are alternately known asanhydro nucleosides. Since the sugar-heterocycle bond of suchcyclonucleosides is "syn" with the heterocycle base, nucleophilicaddition with inversion at this site also yields the desiredribofuranoside configuration of the added nucleotide after addition ofthe second synthon to the first synthon. The linking atom between the 3'position of the sugar and the 2 or 6 position of a pyrimidine base orthe 3' position of the sugar and the 8 position of the purine base canbe oxygen, sulfur or nitrogen.

Since a basic environment is created during coupling of the firstsynthon to the second synthon and an acidic environment (utilizing thepreferred acid-labile phosphate blocking group) is created duringdeblocking of the phosphate blocking group from the nucleotide derivedfrom the second synthon, if blocking groups are utilized on the base orsugar portions of the nucleotides such base or sugar blocking groupsmust be stable to both acidic and basis conditions. Suitable blockinggroups for the heterocyclic base or the sugar are selected to be stableto these conditions. One type of blocking groups that can be used areacid/base stable, hydrogenolysis-sensitive blocking groups; that is,blocking groups which can be removed with molecular hydrogen but notwith acid or base. A benzyl blocking group is such a suitablehydrogenolysis-sensitive blocking group.

Other heterocycle base or sugar blocking groups are those that requiremore pronounced acid or base treatment to de-block than may beexperienced during the basic activation of the nucleophilic displacementreaction of the second synthon blocking group or the acidic removal ofthe phosphate blocking group. Two such blocking groups are the benzoyland isobutyryl groups. Both of these require strong basic conditions fortheir removal. These basic conditions are more stringent than thatrequired to generate the phosphate anion for the nucleophilicdisplacement reaction. This allows the use of such benzoyl or isobutyrylblocking groups for the 6-amino group of adenine, the 2-amino group ofguanine, and the 4-amino group of cytosine. Suitable precursor moleculesfor the second synthons include the xylo derivatives of the commonnucleosides. Certain of these "xylo nucleosides" are commerciallyavailable and others are known in the nucleoside literature.

Xylo nucleosides include but are not limited to xylo derivatives ofadenosine, guanosine, inosine, uridine, cytidine, thymidine,5-methylcytidine, and 2-aminoadenosine, i.e.9-(β-D-xylofuranosyl)adenine, 9-(β-D-xylofuranosyl)guanine,9-(β-D-xylofuranosyl)hypoxanthine, 1-(β-D-xylofuranosyl)uracil,1-(β-D-xylofuranosyl)cytosine, 1-(β-D-xylofuranosyl)thymine,5-methyl-1-(β-D-xylofuranosyl)cytosine, and2-amino-9-(β-D-xylofuranosyl)adenine. They also include the xyloequivalents of the common 2'-deoxy nucleosides such as9-(β-D-2'-deoxy-threo-pentofuranosyl)adenine,9-(β-D-2'-deoxy-threo-pentofuranosyl)guanine,9-(β-D-2'-deoxy-threo-pentofuranosyl)hypoxanthine,1-(β-D-2'-deoxy-threo-pentofuranosyl)uracil,1-(β-D-2'-deoxy-threopentofuranosyl)cytosine,1-(β-D-2'-deoxy-threopentofuranosyl)thymine,5-methyl-1-(β-D-2'-deoxy-threo-pentofuranosyl)cytosine,and 2-amino-9-(β-D-2'-deoxy-threo-pentofuranosyl)adenine.

Other preferred nucleosides that are suitable precursors for the secondsynthon include but are not limited to 2'-fluoro, 2'-methoxy,2'-O-allyl, 2'-methyl, 2'-ethyl, 2'-propyl, 2'-chloro, 2'-iodo,2'-bromo, 2'-amino, 2'-azido, 2'-O-methyl, 2'-Q-ethyl, 2'-Q-propyl,2'-O-nonyl, 2'-Q-pentyl, 2'-O-benzyl, 2'-Q-butyl,2'-Q-(propylphthalimide), 2'-S-methyl, 2'-S-ethyl, 2'-aminononyl,2'-aralkyl, and 2'-alkylheterocyclo such as propylimidazoyl derivativesof the above 2'-deoxy-threo-pentofuranosyl nucleosides. Representativesof this group include but are not limited to9-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)adenine,9-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)guanine,9-(β-D-2'-deoxy-2-fluoro-threo-pentofuranosyl)hypoxanthine,1-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)uracil,1-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)cytosine,1-(β-D-2'-deopxy-2'-fluoro-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)cytosine,2-amino-9-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)adenine,9-(β-D-2'-deoxy-2'-methoxy-threo-pentofuranosyl)adenine,9-(β-D-2'-deoxy-2'-methoxy-threo-pentofuranosyl)guanine,9-(β-D-2'-deoxy-2-methoxy-threo-pentofuranosyl)hypoxanthine,1-(β-D-2'-deoxy-2'-methoxy-threo-pentofuranosyl)uracil,1-(β-D-2'-deoxy-2'-methoxy-threo-pentofuranosyl)cytosine,1-(β-D-2'-deoxy-2'-methoxy-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2'-deoxy-2'-methoxy-threo-pentofuranosyl)cytosine,2-amino-9-(β-D-2'-deoxy-2'-methoxy-threo-pentofuranosyl)adenine,9-(β-D-2'-deoxy-2'-O-allyl-threo-pentofuranosyl)adenine,9-(β-D-2'-deoxy-2'-O-allyl-threo-pentofuranosyl)guanine,9-(β-D-2'-deoxy-2'-O-allyl-threo-pentofuranosyl)hypoxanthine,1-(β-D-2'-deoxy-2'-O-allyl-threo-pentofuranosyl)uracil,1-(β-D-2'-deoxy-2'-O-allyl-threo-pentofuranosyl)cytosine,1-(β-D-2'-deoxy-2'-O-allyl-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2'-deoxy-2'-O-allyl-threo-pentofuranosyl)cytosine,2-amino-9-(β-D-2'-deoxy-2' -O-allyl-threo-pentofuranosyl)-adenine,9-(β-D-2'-deoxy-2'-methyl-threo-pentofuranosyl)-adenine,9-(β-D-2'-deoxy-2'-chloro-threo-pentofuranosyl)-guanine,9-(β-D-2'-deoxy-2-amino-threo-pentofuranosyl)hypoxanthine,1-(β-D-2'-deoxy-2'-O-nonyl-threopentofuranosyl)uracil,1-(β-D-2'-deoxy-2'-O-benzyl-threopentofuranosyl)cytosine,1-(β-D-2'-deoxy-2'-bromo-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2'-deoxy-2'-O-butylthreo-pentofuranosyl)cytosine, and2-amino-9-[β-D-2'-deoxy-2'-O-(propylphthalimide)-threo-pentofuranosyl)adenine.The 2'-deoxy-2'-fluoro-threo-pentofuranosyl, and2'-deoxy-2'-methoxy-threo-pentofuranosyl nucleosides are particularlypreferred in that the 2'-fluoro and 2'-methoxy groups give improvednuclease resistance to oligonucleotide bearing these substituents ontheir respective nucleotides.

Further preferred nucleosides that are suitable precursors for thesecond synthon include but are not limited to thexylofuranosyl or2'-deoxy-threo-pentofuranosyl derivatives of 3-deaza purine and2-substituted amino purine nucleosides including but not limited to3-deaza-2'-deoxyguanosine, 3-deaza-3-nonyl-2'-deoxyguanosine,3-deaza-3-allyl-2'-deoxyguanosine, 3-deaza-3-benzyl-2'-deoxyguanosine,3-deaza-3-nonyl-2'-deoxyguanosine,N2-[imidazol-1-yl-(propyl)]-2'-deoxyguanosine, and2-amino-N2-[imidazol-1-yl(propyl)]adenosine.

Another preferred group of nucleoside precursors for the second synthoninclude the carbocyclic nucleosides, i.e. nucleosides having a methylenegroup in place of the pentofuranosyl ring oxygen atom. Such carbocycliccompounds may exhibit increased stability towards chemical manipulationduring activation of the xylo nucleosides for nucleophilic attack.

The xylo nucleoside or derivatized xylo nucleoside is reacted with asuitable phosphorylating agent to phosphorylate the second synthonprecursor. Various phosphorylation reactions are known in the art suchas those described in Nucleotide Analogs, by Karl Heinz Scheit, JohnWiley & Sons, 1980, Chapter Four--Nucleotides with Modified phosphateGroups and Chapter Six--Methods Of Phosphorylation; Conjugates OfOligonucleotides and Modified Oligonucleotides: A Review Of TheirSynthesis and Properties, Goodchild, J. (1990), Bioconjugate Chemistry,1:165; and Antisense Oligonucleotides: A New Therapeutic Principle,Uhlmann, E. and Peyman, A. (1990), Chemical Reviews, 90:543.

Preferred phosphorylating agents include phosphoryl chlorides. Suitablephosphoryl chlorides include but are not limited to thiophosphorylchloride, t-butoxyphosphoryl chloride, t-butoxy(methyl)phosphorylchloride, t-butoxy(methyl)thiophosphoryl chloride,t-butoxy(methoxy)phosphoryl chloride. Other phosphoryl chlorides mayinclude t-butoxy(N-morpholino)phosphoryl chloride,t-butoxy(ethoxyethylamino)phosphoryl chloride,t-butoxy(methythioxy)phosphoryl chloride, and the like. Suchphosphorylating agents are utilized to yield the correspondingphosphorothioate, phosphoramidate, phosphotriester, alkylphosphonates,and phosphodiester xylo nucleotides.

Even enzymatic phosphorylation is possible, as for example thephosphorylation of 9-(β-D-xylofuranosyl)guanine by nucleosidephosphotransferase from Pseudomonas trifolii as per the procedure ofSuzaki, S., Yamazaki, A. Kamimura, A., Mitsugi, K., and Kumashior, I.(1970), Chem. Pharm. Bull. (Tokyo), 18:172.

1-(β-D-Xylofuranosyl)uracil 5,-phosphate was identified but notseparated from its 3, isomer as reported by Holy, A. and Sorm, F.(1969), Coll. Czech. Chem. Commun., 34:1929. Also,9-(2'-O-benzyl-β-D-xylofuranosyl)adenine 5'-phosphate was obtained as anintermediate by Hubert-Habart, M. and Goodman, L. (1969), Chem. Commun.,740. Removable of the benzyl blocking group would give the desiredunblocked nucleotide.

Additionally, the alkylphosphonates can be prepared by the method ofHoly, A. (1967), Coll. Czech. Chem. Commun., 32:3713. Phosphorothioateshave also been prepared by treatment of the corresponding nucleosidewith trisimidazolyl1-phosphinesulfide followed by acid hydrolysis withaqueous acetic acid, Eckstein, F. (1966 & 1970), J. Am. Chem. Soc.,88:4292 & 92:4718, respectively. A more preferred method is by selectivethiophosphorylation by thiophosphorylchloride in triethylphosphate,Murray, A.W. and Atkinson, M.R. (1968), Biochemistry, 7:4023.

The appropriate phosphorylated xylo nucleotide is then activated fornucleophilic displacement at its 3' position by reacting the 3'-hydroxyl group of the xylo compound with an appropriate anhydride,chloride, bromide, acyloxonium ion, or through an anhydro or cyclonucleoside or the like to convert the 3'-hydroxyl group of the xylonucleoside to an appropriate leaving group.

In a further method of synthesis, treatment of 2',3'-anhydroadenosinewith sodium ethylmercaptide gives9-[3-deoxy-3-(ethylthio)-β-D-xylofuranosyl]adenine. Treatment of thiscompound with a first synthon nucleophile may generate a terminal2-ethylthio arabinofuranosyl nucleoside that could be desulfurized toyield the corresponding 2'-deoxynucleoside.

If during phosphorylation or conversion of the xylo 3'-hydroxyl to a3'-activated leaving group stereospecific diastereomers are notobtained, after completion of the phosphorylation or conversion of the3'-hydroxyl to an activated leaving group, the Rp and Sp diastereomersof these compounds will then be isolated by HPLC. This will yield purediastereomers in a stereospecific form ready for use as the secondsynthons of Scheme 1.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLE 1 Xyloadenosine Method A - Condensation Reaction

Adenine is condensed with 1,2,3,5-tetra-O-acetyl-D-xylopentofuranosidein the presence of TiCL₄ as the condensation catalyst in a polar solventutilizing the method of Szarek, W.A., Ritchie, R.G.S., and Vyas, D.M.(1978), Carbohydr. Res., 62:89.

Method B - Alkylation Reaction

8-Mercaptoadenine is alkylated with5-deoxy-5-iodo-1,2-O-isopropylidine-xylofuranose afollowed by treatmentwith acetic acid/acetic anhydride/sulfuric acid and then ammonia. Thisyields an 8-5'-anhydro intermediate nucleoside that is oxidized withaqueous N-bromosuccinimide to give the sulfoxide. This is blocked withbenzoic anhydride and after a Pummerer rearrangement can be desulfurizedwith Rainey nickel to give 9-β-D-xylofuranosyladenine as per theprocedure of Mizuno, Y., Knaeko, C., Oikawa, Y., Ikeda, T, and Itoh, T.(1972), J. Am. Chem. Soc., 94:4737.

EXAMPLE 2 3-Deaza-9-(β-D-xylofuranosyl)guanine

In a manner similar to Example Method A, 3-deazaguanine is condensedwith 1,23,5-tetra-O-acetyl-D-xylopentofuranoside to yield the titlecompound.

EXAMPLE 3 N⁶ -Benzoyl-9-(2'-Deoxy-2'-fluoro-threo-pentofuranosyl)adenine

In a manner similar to Example 1, Method A, N⁵ -benzoyladenine iscondensed with1,3,5-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yield thetitle compound.

EXAMPLE 4 1-(2'-Deoxy-2'-methoxy-β-D-xylofuranosyl)uridine

In a manner similar to Example 1, Method A, uracil is condensed with1,3,5-tri-O-acetyl-2-deoxy-2-methoxy-D-threopentofuranoside to yield thetitle compound.

EXAMPLE 5 1-(2'-Deoxy-2'-O-allyl-β-D-threo-pentofuranosyl)cytosine

In a manner similar to Example 1, Method A, cytosine is condensed with1,3,5-tri-O-acetyl-2-deoxy-2-O-allyl-D-threo-pentofuranoside to yieldthe title compound.

EXAMPLE 6 Xyloguanosine Method A

In a manner similar to Example 1, Method A, guanine is condensed with1,2,3,5-tetra-O-acetyl-D-xylopentofuranoside to yield the titlecompound.

Method B

The chloromercury derivative of 2-acetamido-6-chloropurine is condensedwith 2,3,5-tri-O-acetyl-β-D-ribofuranosylpurine utilizing the method ofLee et. al. (1971), J. Med. Chem., 14:819. The condensation product wastreated with ammonia to yield2-amino-6-chloro-9-(β-D-xylofuranosyl)purine. Further treatment withsodium hydroxyethylmercaptide gives the title compound.

EXAMPLE 7 2-Amino-6-mercapto-9-(β-D-xylofuranosyl)purine

2-Amino-6-chloro-9-(β-D-xylofuranosyl)purine as prepared by the Example6, Method B, is treated with sodium hydrosulfide to give the titlecompound.

EXAMPLE 8 9-(2'-Deoxy-2'-methyl-D-threo-pentofuranosyl)quanine

In a manner similar to Example 1, Method A, guanine is condensed with1,3,5-tri-O-acetyl-2-deoxy-2-methyl-D-threopentofuranoside to yield thetitle compound.

EXAMPLE 9 2-Amino-xyloadenosine

2-amino-8-mercaptoadenine is treated in the manner as per Example 6,Method B to yield the title compound.

EXAMPLE 10 Carbocyclic Xyloadenosine

5-Amino-4,6-dichloropyrimidine is treated with(±)-4α-amino-2α,3β-dihydroxy-1α-cyclopentanemethanol to give apyrimidine intermediate that is aminated and ring closed to yield thecarbocyclic analog of xylofuranosyladenine as per the procedure ofVince, R. and Daluge, S. (1972), J. Med. Chem., 15:171.

EXAMPLE 11 Carbocyclic Xyloinosine

5-Amino-6-chloro-pyrimidyl-4-one when treated with(±)-4α-amino-2α-,3β-dihydroxy-1α-cyclopentanemethanol will give apyrimidine intermediate that is then aminated and ring closed to yieldthe carbocyclic analog of xylofuranosylinosine as per the procedure ofExample 8.

EXAMPLE 12 O²,3'Cyclothyimidine

3'-O-Mesylthymidine is treated with boiling water and the pH is adjustedto pH 4-5 according to the procedure of Miller, N. and Fox, J.J. (1964),J. Org. Chem., 29:1771 to yield the title compound. This same compoundcan also prepared from 3'-deoxy-3'-iodothymidine by treatment withsilver acetate in acetonitrile.

Method B

O²,3'-Cyclothymidine and other 2'-deoxynucleosides are prepared by thetreatment of the appropriate nucleoside with(2-chloro-1,1,3-trifluoroethyl)diethylamine in dimethylformamideaccording to the procedure of Kowollik, G., Gaertner, K., and Langen, P.(1969), Tetrahedron Lett., 3863.

EXAMPLE 3 O²,3'-Cyclouridine

3'-O-tosyluridine is treated with t-butoxide according to the procedureof Letters, R. and Michelson, A.M. (1961), J. Chem. Soc., 1410. Thiscompound is also prepared by treatment of3'-O-mesyl-2',5'-di-O-trityluridine with sodium benzoate indimethylformamide followed by detritlation with hydrochloric inchloroform.

EXAMPLE 14 S², 3'-Cyclo-2-thiothymidine

3'-O-mesyl-0²,5'-cyclothymidine is subjected to methanolysis followed bysulfhydryl ion attack. The S²,3'-cyclo linkage is then opened up withbase to yield 2',3'-dideoxy-3'-mercapto-1-(β-D-xylofuranosyl)thymidineas per the procedure of Wempen, I. and Fox, J.J. (1969), J. Org. Chem.,4:1020.

Method B

S²,3'-Cyclo-2-thiouridine is also prepared from 2-thiouridine by themethod of Doerr, I.L. and Fox, J.J. (1967), J. Am. Chem., 89:1760.

EXAMPLE 15 N⁶,5'-Cyclothymidine

5'-O-Trityl-3'-O-mesylthymidine is treated with sodium azide to yieldN⁶,5'-cyclothymidine as one of the products.5'-O-trityl-3'-O-mesylthymidine is also cyclizable toO²,3'-cyclothymidine.

EXAMPLE 6 8,3'-Cycloadenosine

The anhydro ring from the 3' position of the sugar to the 8 position ofthe purine ring is formed by treatment of 5'-O-acetyl-8-bromo-2' (or3')-O-p-toluenesulfonyladenosine with thiourea to yield the8,3'-thiocyclonucleoside (as well as the corresponding 8,2') product asper the procedure of Ikehara, M. and Kaneko, M. (1970), Tetrahedron,26:4251.

EXAMPLE 17 8,3'-Cycloguanosine

The title compound is prepared as per Example 16 utilizing8-bromoguanosine. Both this compound and the compound of Example 16 canbe oxidized to their corresponding sulfoxides via tert-butylhypochlorite in methanol or treated with chlorine in methanolic hydrogenchloride to yield the 3'-sulfo-8-chloro analog in a procedure analogouswith that of Mizuno, Y., Kaneko, O., and Oikawa, Y. (1974), J. Org.Chem., 39:1440.

EXAMPLE 18

1-3'-Bromo-3'-deoxy-N⁴, 2', 5'-O-triacetylxylofuranosyl)cytosine

The title compound is prepared by treating N⁴ -acetylcytidine withacetyl bromide according to the procedure of Marumoto, R. and Honjo, M.(1974), Chem. Pharm. Bull., 22:128.

EXAMPLE 9 9-(3-Deoxy-3-fluoro-β-D-xylofuranosyl)adenine

Treatment of 9-(2,3-anhydro-5-O-benzoyl-β-D-ribofuranosyl)-N,N-dibenzoyl(or N-pivaloyl)adenine with tetraethylammonium fluoride in hotacetonitrile followed by deacylation with sodium methoxide yields9-(3-deoxy-3-fluoro-β-D-xylofuranosyl)adenine as per the procedure ofLichtenthaler, F. W., Emig, P., and Bommer, D. (1969), Chem. Ber.,102:964.

EXAMPLE 20 9-(3-Deoxy-3-fluoro-β-D-xylofuranosyl)guanine

In a like manner to Example 18 the corresponding guanosine compound willbe prepared from the corresponding 2,3-anhydro guanosine.

EXAMPLE 21 9-(3'-Chloro-3'-deoxy-β-D-xylofuranosyl)hypoxanthine

5'-O-Acetylinosine is treated with triphenylphosphine and carbontetrachloride to yield the title compound according to the procedure ofHaga, K., Yoshikawa, M., and Kato, T. (1970), Bull. Chem. Soc. Jpn.,43:3992.

EXAMPLE 229-(2-O-Acetyl-3-chloro-3-deoxy-5-O-pivaloyl-β-D-xylofuranosyl)-6-pivalamidopurine

The title compound is prepared via an intermediate 2',3'-O-acyloxoniumion utilized to introduce a halogen atom at the 3' position and convertthe ribo configuration of a nucleoside into the corresponding3'-halo-3'-deoxy xylo nucleoside. The acyloxonium ion is generated insitu by treatment of 2',3'-O-methoxyethylidineadenosine with pivaloylchloride in hot pyridine. Attack by chloride gives the title compound.Hypoxanthine and guanine nucleoside react in a similar manner. Sodiumiodide will be used to generate he corresponding 3'-iodides according tothe procedure of Robins, M.J., Fouron, Y., and Mengel, R. (1974 , J.Org. Chem., 39:1564.

EXAMPLE 23 9-(2,5,-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine

This compound is prepared by a in situ acyloxonium ion generated bytreating 3',5'-di-O-acetyladenosine with boron trifluoride etheratefollowed by phosphorus tribromide according to the procedure of Kondo,K., Adachi, T., and Inoue, I. (1977), J. Org. Chem., 42:3967. The titlecompound can also be formed by treating adenosine withtetraacetoxysilane and phosphorus tribromide in the presence of borontrifluoride etherate.

EXAMPLE 24 1-(β-D-2'-Deoxy-2'-fluoro-threo-pentofuranosyl)uracil

In a manner similar to Example 3, uracil is condensed with1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threopentofuranoside to yield thetitle compound.

EXAMPLE 25 1-(β-D-2'-Deoxy-2'-fluoro-threo-pentofuranosyl)guanine

In a manner similar to Example 3, guanine is condensed with1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threopentofuranoside to yield thetitle compound.

EXAMPLE 26 1-(β-D-2'Deoxy-2'-fluoro-threo-pentofuranosyl)cytosine

In a manner similar to Example 3, cytosine is condensed with1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threopentofuranoside to yield thetitle compound.

EXAMPLE 27 O²,3'-Cyclo-2'-deoxyoytidine

The title compound is prepared by heating the 3'-O-sulfamate as per theprocedure of Schuman, D., Robins, M.J., and Robins, R.K. (1970), J. Am.Chem. Soc , 92:3434.

EXAMPLE 28 Sp and Rp Xyloadenosine 5'-Monophosphate

N⁶ -Benzoyl-xyloadenosine is phosphorylated with phosphoryl chloride inpyridine and acetonitrile at 0° C. The reaction will be quenched withice water, rendered basic and added to an activated charcoal column.After elution with ethanol/water/concentrated ammonium hydroxide thesolvent is evaporated to dryness and the residue dissolved in water andpassed through an ion exchange column. The benzoyl blocking group isremoved in concentrated ammonium hydroxide followed by separation of thediastereomers by HPLC to yield the title compound.

EXAMPLE 29 Sp and Rp 1-(β-D-2'-Deoxy-2'-fluoro-threopentofuranosyl)uracil5'-t-butoxy(methyl)phosphonate

1-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)thymine will bephosphorylated with t-butoxy(methyl)phosphoryl chloride intrimethylphosphate at 0° C. for 3 hrs. The solution is added to coldanhydrous ether. The racemic precipitate is taken up in acetonitrile andthe Sp and Rp diastereomers of the title compound separated by HPLCutilizing a gradient of acetonitrile and triethylammonium acetatebuffer.

EXAMPLE 30 Sp and Rp1-(β-D-2'-Deoxy-2'-fluoro-threopentofuranosyl)cytosine5'-t-butoxy(methyl)phosphonate

1-(β-D-2'-deoxy-2'-fluoro-threo-pentofuranosyl)cytosine will bephosphorylated and purified as per the procedure of Example 29 to givethe diastereomers of the title compound.

EXAMPLE 31 Sp and Rp N⁵-Benzoyl-9-(β-D-2'-Deoxy-2'-fluoro-threopentofuranosyl)adenine5'-t-butoxy(methyl)phosphonate

N⁶ -Benzoyl-9-(β-D-2'-deoxy-2'-fluoro-threopentofuranosyl)adenine willbe phosphorylated and purified as per the procedure of Example 29 togive the diastereomers of the title compound.

EXAMPLE 32 Sp and Rp 9-(β-D-2'-Deoxy-2'-fluoro-threopentofuranosyl)guanine5'-t-butoxy(methyl)phosphonate

9-(β-D-2'-Deoxy-2'-fluoro-threo-pentofuranosyl)guanine will bephosphorylated and purified as per the procedure of Example 29 to givethe diastereomers of the title compound.

EXAMPLE 33 Sp and Rp Xylofuranosyluracil 5'-t-butoxyphosphorothioate

Xylofuranosyluracil will be phosphorothioated Witht-butoxythiophosphorylchloride in triethylphosphate utilizing the methodof Murray, A.W. and Atkinson, M.R. (1968), Biochemistry, 7:4023. Thediastereomers of the title compound are separated by HPLC.

EXAMPLE 34 Sp and Rp9-(2'-Deoxy-2'-methyl-β-D-threopentofuranosyl)guanine5'-Methylphosphonate

9-(2'-Deoxy-2'-methyl-β-D-threo-pentofuranosyl)guanine will bealkylphosphonated utilizing the procedure of Holy, A. (1967), Coll.Czech. Chem Commun., 32:3713. The racemic phosphorylation product isseparated into its Sp and Rp diastereomers using HPLC chromatography toyield the title compound.

EXAMPLE 35 Sp and Rp 9-(2'-Deoxy-β-D-threo-pentofuranosyl)hypoxanthine5'-Phosphormorpholidate

9-(2'-Deoxy-β-D-threo-pentofuranosyl)hypoxanthine is phosphorylatedaccording to the procedure of Example 28. The resulting 5'-phosphateintermediate will be phosphormorpholidated by treatment with activatedwith dicyclohexylcarbodiimide in the presence of morpholine according tothe procedure of Moffatt, J.G. and Khorana, H.G. (1961 ), J. Am. Chem.Soc., 83:3752 to yield the racemic title compound. The diastereomers ofthe product are separated by HPLC.

EXAMPLE 36 Sp nd Rp9-(2'-Deoxy-2'-O-allyl-β-D-threopentofuranosyl)cytosine 5'-Phosphate

9-(2'-Deoxy-2'-allyl-β-D-threo-pentofuranosyl)cytosine will bephosphorylated according to the procedure of Example 28 to yield theracemic title compound. The diastereomers are separated by HPLC.

EXAMPLE 37 Sp and Rp 9-(2'-Deoxy-2'-methoxy-β-D-threo-pentofuranosyl)uracil 5'-Phosphate

9-(2'-Deoxy-2'-methoxy-β-D-threo-pentofuranosyl)uracil will bephosphorylated according to the procedure of Example 28 to yield theracemic title compound. The diastereomers are separated by HPLC.

EXAMPLE 38 Sp and Rp 3-Deaza-9-(xylofuranosyl)guanine 5'-Phosphate

3-Deaza-9-(xylofuranosyl)guanine Will be phosphorylated according to theprocedure of Example 28 to yield the racemic title compound. Thediastereomers are separated by HPLC.

EXAMPLE 39 Sp and Rp Xyloguanosine 5'-Phosphorothioate

Xyloguanosine will be phosphorothioated with thiophosphoryl chlorideaccording to the procedure of Example 28 to yield the racemic titlecompound. The diastereomers are separated by HPLC.

EXAMPLE 40 Sp and Rp Carbocyclic Xyloadenosine 5'-Phosphate

In a like manner to Example 28, carbocyclic xyloadenosine will betreated with phosphoryl chloride to yield the racemic title compound.The diastereomers will be separated by HPLC.

EXAMPLE 41 Activated 3'-Deoxy-3'-Active Leaving Group PhosphorylatedNucleosides

The 3'-halo nucleotides can be treated with methoxide to give anunstable 2',3'-anhydro intermediate that slowly forms the corresponding3,3'-cyclonucleoside. The cyclonucleoside in turn can undergonucleophilic attack to yield other 3'-deoxy-3'-substituted derivatives,as for instance, the tosyl, triflate, trichloroacetimidate or otheractive species.

EXAMPLE 42 N⁶-Benzoyl-9-(3'-Deoxy-3'-tosyl-2'-deoxy-2'-fluoro-β-D-threopentofuranosyl)adenine5'-Rp t-Butoxy(methyl)phosphonate

N⁶ -Benzoyl-9-(2'-deoxy-2'-fluoro-β-D-threo-pentofuranosyl)adenine 5'-Rpt-butoxy(methyl)phosphonate will be treated withp-toluenesulfonylchloride in pyridine as per the procedure of Reist,E.J., Bartuska, V.J., Calkins, D.F., and Goodman, L. (1965), J. Org.Chem., 30:3401 to yield the title compound.

EXAMPLE 439-(3'-Deoxy-3'-tosyl-2'-deoxy-2'-methoxy-β-D-threopentofuranosyl)uracil5'-Rp t-Butoxy(methyl)phosphonate

9-(2'-Deoxy-2'-methoxy-β-D-threo-pentofuranosyl)uridine 5'-Rpt-butoxy(methyl)phosphonate will be treated withp-toluenesulfonylchloride in pyridine according to the procedure ofExample 42 to yield the title compound.

EXAMPLE 449-(3'-Deoxy-3'-tosyl-2'-deoxy-2'-fluoro-β-D-threopentofuranosyl)uracil5'-Rp t-Butoxy(methyl)phosphonate

9-(2'-Deoxy-2'-fluoro-β-D-threo-pentofuranosyl)uridine 5'-Rpt-butoxy(methyl)phosphonate will be treated withp-toluenesulfonylchloride in pyridine according to the procedure ofExample 42 to yield the title compound.

EXAMPLE 459-(3'-Deoxy-3'-tosyl-2'-deoxy-2'-fluoro-β-D-threopentofuranosyl)cytosine5'-Rp t-Butoxy(methyl)phosphonate

9-(2'-Deoxy-2'-fluoro-β-D-threo-pentofuranosyl)cytosine 5'-Rpt-butoxy(methyl)phosphonate will be treated withp-toluenesulfonlychloride in pyridine according to the procedure ofExample 42 to yield the title compound.

EXAMPLE 469-(3'-Deoxy-3'-tosyl-2'-deoxy-2'-fluoro-β-D-threo-pentofuranosyl)guanine5'-Phosphate

9-(2'-Deoxy-2'-fluoro-β-D-threo-pentofuranosyl)guanine 5'-Sp phosphatewill be treated with p-toluenesulfonyl chloride in pyridine according tothe procedure of Example 42 to yield the title compound.

EXAMPLE 479-(3'-Deoxy-3'-tosyl-2'-deoxy-2'-O-allyl-β-D-threopentofuranosyl)thymine5'-Phosphate

9-(2'-deoxy-2'-O-allyl-β-D-threo-pentofuranosyl)thymine 5'-Rp phosphatewill be treated with p-toluenesulfonyl chloride in pyridine according tothe procedure of Example 42 to yield the title compound.

EXAMPLE 48 3'-Deoxy-3'-trifluoromethanesulfonylxyloguanosine5'-Phosphorothioate

Xyloguanosine 5'-Sp phosphorothioate will be treated withtrifluoromethane sulfonic acid anhydride in the presence of a sodiumhydride to yield the title compound.

EXAMPLE 49 Carbocyclic 3'-Deoxy-3'-trifluoromethanesulfonylxyloadenosine5'-Phosphate

In a like manner to Example 48, carbocyclic xyloadenosine 5'-Rpphosphate will be treated with trifluoromethane sulfonic acid to yieldthe title compound.

EXAMPLE 50 S²,3'-Cyclo-2-thiothymidine

S²,3'-Cyclo-2-thiothymidine is prepared from3'-O-mesyl-O²,5'-cyclothymidine via methanolysis followed by sulfhydrylion attack. The S² 2,3'-cyclo linkage is then opened up with base toyield 2',3'-dideoxy-3'-mercapto-1-(β-D-xylofuranosyl)thymidine, Wempen,I. and Fox, J.J. (1969), J. Org. Chem., 34:1020. The 3' position willthen be activated to nucleophilic attack via an active leaving groupsuch as conversion of the mercapto to a tosyl leaving group. In a likemanner S²,3'-Cyclo-2-thiouridine prepared from 2-thiouridine by themethod of Doerr, I.L. and Fox, J.J. (1967), J. Am. Chem., 89:1760, canbe ring opened and then derivatized with an activated leaving group suchas a tosylate.

EXAMPLE 51 Synthesis of 2'-Deoxy-2'-fluoro substituted CGA CTA TGC AACTAC Rp Methylphosphonate Linked Oligonucleotide

1-(2'-Fluoro-2'-deoxy-β-D-ribofuranosyl)cytosine 5'-Rp methylphosphonatewill be attached via its 3' hydroxyl to CPG beads in a standard manneras practiced in automated nucleic acid synthesis. This nucleotide formsthe first synthon 1

a) Activation of Synthon 1

The beads are washed with acetonitrile and treated With 1.1 equivalentsof sodium hydride in acetonitrile to form an anion on themethylphosphonate moiety.

(b) Addition of Synthon 2 and Coupling of Synthons 1 and 2

2.0 Equivalents of N⁶-Benzoyl-9-(3'-deoxy-3'-tosyl-2'-deoxy-2'-fluoro-β-D-threo-pentofuranosyl)adenine5'-Rp t-butoxy(methyl)phosphonate in acetonitrile is added withstirring. After completion of the nucleophilic reaction and formation ofthe cytosine-adenine dimer as judged by tlc, the beads are filtered andwashed with acetonitrile.

c) Removal of t-Butoxy Blocking Group

The beads are re-suspended in acetonitrile and 1.5 equivalents oftrichloracetic acid is added. The reaction is stirred to remove thet-butoxy blocking group on the terminal 5'-methylphosphonate group ofthe adenosine nucleotide, followed by washing with acetonitrile.

(d) Cycling

The reaction is cycled to step (a), followed by addition of the nextsynthon 2 nucleotide at step (b), and deblocking at step (c). Thereaction is further cycled for each nucleotide making up the specificsequence of the oligonucleotide. After the addition of the penultimatenucleotide its phosphate moiety is activated at step (a) and the finalnucleosidic unit is added at step (b) as a xylofuranosyl nucleoside. Theoligonucleotide is concurrently deblocked and removed from the CPG beadsby treatment with concentrate ammonium hydroxide.

What is claimed is:
 1. A compound having the structure: ##STR10##wherein: Q is O or CH₂ ;R_(D) is O, S, methyl, O--C₁ -C₁₂ -alkyl, S--C₁-C₁₂ -alkyl, amino or substituted amino, where said substituent is C₁-C₁₂ -alkyl or C₆ -C₁₄ -aryl; R_(E) is O or S; R_(F) is H or a blockinggroup; R_(X) is H, OH, C₁ -C₁₂ -alkyl, substituted C₁ -C₁₂ -alkyl, F,Cl, Br, OCF₃, O--C₁ -C₁₂ -alkyl, O-substituted C₁ -C₁₂ -alkyl, OCH₂CH═CH₂, OCH₂ CCH, and C(O)--C₁ -C₁₂ -alkyl, wherein said substituent isC₁ -C₁₂ -haloalkyl, C₁ -C₁₂ -alkenyl, C₁ -C₁₂ -alkoxy, C₁ -C₁₂-thioalkoxy, C₁ -C₁₂ -haloalkoxy, C₆ -C₁₄ -aryl, halogen, hydroxyl,amino, azido, carboxy, cyano, nitro, or mercapto; B_(X) is a nucleosidebase or a blocked nucleoside base; and L is a leaving group or togetherL and Bx are a C2 or C6 pyrimidine-3'xylo cyclo-nucleoside or C8purine-3'xylo cyclo-nucleoside.
 2. The compound of claim 1 wherein L isselected from the group consisting of halogen, C₁ -C₁₂ -alkyl-sulfonyl,substituted C₁ -C₁₂ -alkylsulfonyl, C₆ -C₁₄ -arylsulfonyl, substitutedC₆ -C₁₄ -arylsulfonyl, trichloroacetimidate, and C(O)-C₁ -C₁₂ -alkyl,wherein said substituent is halogen, hydroxyl, amino, azido, carboxy,cyano, or nitro.
 3. The compound of claim 1 wherein L is selected fromthe group consisting of chloro, fluoro, bromo, iodo,p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl(mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl,trifluoromethylsulfonyl (triflate), trichloroacetimidate,2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl and2,4,6-trichlorophenyl.
 4. The compound of claim 1 wherein R_(F) isselected from the group consisting of H, t-butyl, dimethoxytrityl ortetrahydropyranyl.
 5. The compound of claim 1 wherein B_(X) is apyrimidinyl-1 or purinyl-9 moiety.
 6. The compound of claim 1 whereinB_(X) is adenine, guanine, hypoxanthine, uracil, thymine, cytosine,2-aminoadenine or 5-methylcytosine.
 7. The compound of claim 1 wherein Qis O.
 8. The compound of claim 1 having Sp stereochemistry.
 9. Thecompound of claim 1 having Rp stereochemistry.